Metabolic Syndrome-Improving Agent and Medicine, Supplement, Functional Food and Food Additive Containing the Same

ABSTRACT

A metabolic syndrome relieving agent that is free from a problem of side effects and can be taken for a long term is provided. Aurapten is used as an agent for relieving a metabolic syndrome. Since aurapten has functions of activating PPAR α  and PPARγ, promoting the secretion of adiponectin in adipocytes and inhibiting the generation of VLDLs in hepatic cells, it is possible to prevent or treat diseases such as insulin resistance, hyperinsulinism, type 2 diabetes, obesity, visceral fat obesity, hypertension, hyperlipemia, arteriosclerosis and the like and thus prevent or treat the metabolic syndrome. Also, as understood from the fact that citrus fruits such as a hassaku orange, a sweet summer orange or the like containing aurapten have been eaten for many years, they have no problems in terms of safety and have a low calorie content, and therefore, they can be taken for a long term. Further, since aurapten is tasteless and odorless, it does not impair the unique taste of a food when added to this food, so that it can be added to foods and taken.

TECHNICAL FIELD

The present invention relates to a metabolic syndrome relieving agent,and drugs, supplements, functional foods and food additives containingthe same.

BACKGROUND ART

For the past several years, the concept of metabolic syndrome has beenexpounded by WHO (the World Health Organization) as well as the NationalCholesterol Education Program (NCEP) in the United States. The metabolicsyndrome is the state in which various diseases causingarteriosclerosis, for example, visceral fat obesity, insulin resistance,diabetes, hyperlipemia, hypertension, etc. cluster together, so thatangina pectoris, myocardial infarction or the like occurs easily. It isconsidered that the metabolic syndrome is ascribable to visceraladiposity and hypertrophic adipocytes.

The following two mechanisms have been revealed as how the visceraladiposity is associated with the development of diabetes, hyperlipemia,hypertension or the like. One is the mechanism in which a large amountof glyceride accumulated in visceral fat is decomposed in the fastingstate, and a large amount of free fatty acid and glycerol, which areproducts of the decomposition, are released and flow in excess into theliver, leading to hyperlipemia, hyperglycemia and hyperinsulinism. Theother is the mechanism in which the visceral adiposity causes abnormaladypocytokine secretion, which inhibits the secretion of adiponectin,for example, so that diabetes, arteriosclerosis or the like occurs (forexample, see Non patent document 1). It has been revealed that theadiponectin activates a peroxisome proliferator-activated receptor(PPAR) α and AMP kinase so as to promote fatty acid burning or the like,thus decreasing the content of neutral fats in tissue and thereby, forexample, relieving the insulin resistance or the like. Further, therehas been a report that, besides the above, the adiponectin hasantidiabetic, anti-arteriosclerotic, antihypertensive andanti-inflammatory effects.

On the other hand, PPARs, which are intranuclear receptors, are said tobe associated with the relief of insulin resistance, hyperinsulinism,type 2 diabetes as well as obesity, hypertension, hyperlipemia andarteriosclerosis. PPARs are known to have three types, i.e., α, δ and γ,and several subtypes. PPAR_(α) is expressed mainly in the hepatic cellsand also in other cells such as myocardial cells and gastrointestinalcells, and concerned with fatty acid oxidation, ketogenesis andapolipoprotein generation. Although PPARδ is not considered to havetissue specificity and is expressed throughout the body, it is expressednotably in large intestinal cancer cells. PPARγ can be classified intotwo subtypes, i.e., type γ1 and type γ2. The type γ1 is expressed inadipose tissues, immune system tissues, the adrenal gland and the smallintestine, whereas the type γ2 is expressed specifically in adipocytesand plays an important role in differentiation induction of theadipocytes and fat synthesis.

The number of patients afflicted with the metabolic syndrome tends toincrease mainly in developed countries. Accordingly, there is an urgentneed for a metabolic syndrome relieving agent that has excellent safetyand can be taken over a long term.

Non patent document 1: Yuji MATSUZAWA, “Concept of Metabolic Syndromeand Molecular Mechanism,” The Journal of Therapy, November 2004, Vol.86, No. 11, pages 011-016. DISCLOSURE OF INVENTION Problem to be Solvedby the Invention

It is an object of the present invention to provide a metabolic syndromerelieving agent that is free from a problem of side effects and can betaken for a long term.

Means for Solving Problem

In order to achieve the above-mentioned object, an agent for relieving ametabolic syndrome according to the present invention contains aurapten.The metabolic syndrome includes diseases, for example, insulinresistance, hyperinsulinism, type 2 diabetes, hyperlipemia,arteriosclerosis, hypertension, obesity, visceral fat obesity and thelike.

EFFECTS OF THE INVENTION

From the viewpoints of side effects and long-term intake, the inventorof the present invention conducted a series of studies mainly onsubstances contained in food. In the course of the studies, the inventorfound that aurapten contained in citrus fruits had functions ofactivating PPAR_(α) and PPARγ, promoting the secretion of adiponectin inadipocytes and inhibiting the generation of very low-densitylipoproteins (VLDLs) in hepatic cells, thus arriving at the presentinvention. In other words, according to the metabolic syndrome relievingagent of the present invention, since the PPAR activation promotes fatburning so as to inhibit the secretion of TNFα and free fatty acid, thestate of adipocytes can be normalized, thus making it possible toprevent or treat diseases, for example, insulin resistance,hyperinsulinism, type 2 diabetes, obesity, visceral fat obesity,hypertension, hyperlipemia, arteriosclerosis and the like. Also, themetabolic syndrome relieving agent according to the present inventionpromotes the secretion of adiponectin in adipocytes so as to promotefatty acid burning and PPARα activation, thereby normalizing the stateof adipocytes, and suppresses endangiitis or the like, therebypreventing LDLs from being taken into blood vessels, for example.Consequently, this also makes it possible to prevent or treat diseases,for example, insulin resistance, hyperinsulinism, type 2 diabetes,obesity, visceral fat obesity, hypertension, hyperlipemia,arteriosclerosis and the like. Further, since the metabolic syndromerelieving agent according to the present invention inhibits thegeneration of VLDLs in hepatic cells so as to suppress an increase inneutral fats, it is possible to prevent or treat diseases, for example,hyperlipemia and the like. In this way, since the metabolic syndromerelieving agent according to the present invention can prevent or treatthe above-described diseases, for example, by being administered to ahuman and mammals other than the human, it is considered to have anexcellent effect of relieving a metabolic syndrome.

Moreover, citrus fruits, for example, a hassaku orange, a sweet summerorange, a Watson pomelo and a grapefruit, containing a large amount ofaurapten have been eaten for many years and confirmed in terms ofsafety. Also, aurapten has a low calorie content and, in this regard,does not cause any problem even if it is taken by a diabetic patient, anobese patient or the like for a long term. Further, since aurapten istasteless and odorless, it does not impair the unique taste of a food orthe like when added to this food, so that it can be added to foods andtaken daily over a long term, for example.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing a PPARγ ligand activity of aurapten in anexample of the present invention.

FIG. 2 is a graph showing a PPAR_(α) ligand activity of aurapten inanother example of the present invention.

FIG. 3 is a graph showing adiponectin mRNA expression amounts byaurapten in yet another example of the present invention.

FIG. 4. a graph showing an adiponectin secretion promoting effect ofaurapten in the above-noted example of the present invention.

FIG. 5 is a graph showing a VLDL secretion inhibiting effect by auraptenin yet another example of the present invention.

DESCRIPTION OF THE INVENTION

The metabolic syndrome relieving agent according to the presentinvention has the functions of activating a PPAR, promoting thesecretion of adiponectin in adipocytes, inhibiting the generation ofVLDLs in hepatic cells, inhibiting the secretion of TNFα and free fattyacid in adipocytes and promoting β oxidation of fat in hepatic cells,for example. The PPAR to be activated is at least one of PPAR_(α) andPPARγ, for example, and preferably is both of them. Further, themetabolic syndrome relieving agent according to the present inventioninduces at least one of apoptosis, differentiation and shrinkage of anadipocyte, for example. It should be noted that the metabolic syndromerelieving agent according to the present invention may contain variousadditives other than aurapten, and further may contain other componentshaving a PPAR activating function, for example.

In the metabolic syndrome relieving agent according to the presentinvention, the aurapten to be used is not particularly limited, andexamples thereof include those derived from citrus fruits. Inparticular, aurapten derived from a fruit juice, that derived from afruit and that derived from a peel are preferable, and only one of or acombination of two or more of them may be used. Examples of the citrusfruits can include a sweet summer orange, a hassaku orange, a Watsonpomelo, a grapefruit and the like. The aurapten may be a productobtained by isolation and purification from the above-noted citrusfruits or may be a commercially available product, for example.

Next, a drug according to the present invention is a drug for preventingor treating a metabolic syndrome, and the drug contains the metabolicsyndrome relieving agent according to the present invention. The drugaccording to the present invention is administered to a human andmammals other than the human, thereby making it possible to prevent ortreat diseases, for example, insulin resistance, hyperinsulinism, type 2diabetes, hyperlipemia, arteriosclerosis, hypertension, obesity andvisceral fat obesity. The drug of the present invention may contain notonly the metabolic syndrome relieving agent according to the presentinvention but also other components having a PPAR activating functionand pharmaceutically acceptable additives, for example. In the drugaccording to the present invention, examples of its specific dosage formcan include tablets, fine grains (including pulvis), capsules, solution(including syrup) and the like. The drug according to the presentinvention can be manufactured by using an additive or a base, etc. thatis suitable for the respective dosage form as necessary according to aregular method described in the Pharmacopoeia of Japan or the like.Also, a route of administration is not particularly limited but can be,for example, an oral administration or a parenteral administration.Examples of the parenteral administration can include intraoraladministration, tracheobronchial administration, intrarectaladministration, subcutaneous administration, intramuscularadministration, intravenous administration and the like.

Now, a supplement according to the present invention is a supplement forpreventing or relieving a metabolic syndrome, and the supplementcontains the metabolic syndrome relieving agent according to the presentinvention. The supplement according to the present invention is taken bya human and mammals other than the human, thereby making it possible toprevent or relieve diseases, for example, insulin resistance,hyperinsulinism, type 2 diabetes, hyperlipemia, arteriosclerosis,hypertension, obesity and visceral fat obesity. The supplement of thepresent invention may contain not only the above-mentioned metabolicsyndrome relieving agent according to the present invention but alsovarious additives, other supplements and the like, for example, othercomponents having a PPAR activating function, various vitamins such asvitamin C, amino acids and oligosaccharides. The supplement according tothe present invention may be in any form without particular limitation,which can be, for example, tablets, fine grains (including pulvis),capsules, solution (including syrup) or the like.

Next, a functional food according to the present invention is afunctional food for preventing or relieving a metabolic syndrome, andthe functional food contains the metabolic syndrome relieving agentaccording to the present invention. The functional food according to thepresent invention is taken by a human and mammals other than the human,thereby making it possible to prevent or relieve diseases, for example,insulin resistance, hyperinsulinism, type 2 diabetes, hyperlipemia,arteriosclerosis, hypertension, obesity and visceral fat obesity. Thefunctional food of the present invention may contain not only theabove-mentioned metabolic syndrome relieving agent according to thepresent invention but also various additives and the like and maycontain, for example, other components having a PPAR activatingfunction. Incidentally, the functional food according to the presentinvention may be in any form without particular limitation, which canbe, for example, noodles, confectionery, functional drinks or the like.

Now, a food additive according to the present invention is a foodadditive for preventing or relieving a metabolic syndrome, and the foodadditive contains the metabolic syndrome relieving agent according tothe present invention. The food additive according to the presentinvention is taken by a human and mammals other than the human, therebymaking it possible to prevent or relieve diseases, for example, insulinresistance, hyperinsulinism, type 2 diabetes, hyperlipemia,arteriosclerosis, hypertension, obesity and visceral fat obesity. Thefood additive of the present invention may contain not only theabove-mentioned metabolic syndrome relieving agent according to thepresent invention but also various additives and the like and maycontain, for example, other components having a PPAR activatingfunction. The food additive according to the present invention may be inany form without particular limitation, which can be, for example,liquid, paste, powder, flakes, granule or the like. Moreover, the foodadditive according to the present invention includes, for example, foodadditives for drinks.

Next, a PPAR activator according to the present invention containsaurapten. The PPAR activator according to the present invention also maycontain components other than aurapten. Examples of the above-notedother components include various additives, other PPAR activators andthe like. The aurapten that can be used in the PPAR activator accordingto the present invention is similar to that in the above-describedmetabolic syndrome relieving agent of the present invention.

The PPAR activator according to the present invention can be used forrelieving the metabolic syndrome and treating a dermatosis or the like,for example. Examples of the dermatosis include the skin of prematureinfants of gestational age less than 33 weeks; atopic and seborrheicdermatitis; inflammation to mucous membranes, such as cheilitis, chappedlips, nasal irritation and vulvovaginitis; eczematous dermatitisresulting from allergic and irritant contact, eczema craquelee,radiation and stasis dermatitis; ulcers and erosions due to chemical orthermal burns, bullous disorders or vascular compromise or ischemiaincluding venous, arterial, embolic or diabetic ulcers; ichthyoses, withor without an associated barrier abnormality; epidermolysis bullosa;psoriasis; hypertrophic scars and keloids; intrinsic aging anddermatoheliosus; mechanical friction blistering; corticosteroid atrophy;and melanoma and non-melanoma skin cancer, including lignin melanoma,basal cell carcinoma, squamous cell carcinoma, actinic keratoses, andvirally induced neoplasia (warts and condylomata accuminata). When thePPAR activator according to the present invention is used for treating adermatosis, it may be in any form without particular limitation, whichcan be, for example, a lotion, a solution, a gel, a cream, an emollientcream, an ointment, a spray or other forms that can be applied locally.

Next, an adiponectin secretagogue according to the present inventioncontains aurapten. The adiponectin secretagogue according to the presentinvention may contain components other than aurapten. The aurapten thatcan be used in the adiponectin secretagogue according to the presentinvention is similar to that in the above-described metabolic syndromerelieving agent of the present invention.

The adiponectin secretagogue according to the present invention can beutilized for relieving the metabolic syndrome, treating a chronic liverdisease such as chronic hepatitis, or the like, for example. This isbecause the adiponectin secretagogue according to the present inventioncan inhibit liver fibrosis in a chronic liver disease such as chronichepatitis, for example. The adiponectin secretagogue according to thepresent invention can be in any form without particular limitation,which can be, for example, drugs, supplements, functional foods or foodadditives.

Now, a use according to the present invention is a use of aurapten formanufacturing a metabolic syndrome relieving agent. Another useaccording to the present invention is a use including administeringaurapten to a human and mammals other than the human for relieving ametabolic syndrome. Yet another use according to the present inventionis a use of aurapten for manufacturing a PPAR activator. The auraptencan be similar to that in the above-described metabolic syndromerelieving agent of the present invention. Examples of the above-notedmammals include a mouse, a rat, a rabbit, a dog, a cat, a cow, a horse,a swine, a monkey and the like.

Now, a method for relieving a metabolic syndrome according to thepresent invention is a method including administering aurapten to ahuman and mammals other than the human. The aurapten that can be used inthe relieving method according to the present invention is similar tothat in the above-described metabolic syndrome relieving agent of thepresent invention. In the relieving method according to the presentinvention, the aurapten to be administered can be in any form withoutparticular limitation, which can be, for example, tablets, fine grains(including pulvis), capsules, solution (including syrup) and the like.Also, a method of administration is not particularly limited but can be,for example, an oral administration or a parenteral administration.Examples of the parenteral administration can include intraoraladministration, tracheobronchial administration, intrarectaladministration, subcutaneous administration, intramuscularadministration, intravenous administration and the like.

Next, a PPAR activating method according to the present invention is amethod including activating a PPAR with aurapten. In the PPAR activatingmethod according to the present invention, it is preferable that thePPAR is activated by bringing the aurapten into contact with anadipocyte or the like, for example. The aurapten that can be used in theactivating method according to the present invention is similar to thatin the above-described metabolic syndrome relieving agent of the presentinvention.

Now, it is preferable that the aurapten in the present invention ismanufactured from a material such as citrus fruits as described earlier.The following is a description of an example of this manufacturingmethod (JP 11 (1999)-29565 A).

First, a peel of a citrus fruit is soaked in water at room temperatureand then centrifuged, thus obtaining a peel oil. Although the peel maybe replaced by a fruit, a pulp or a fruit juice, the use of the peel ispreferable because of the large aurapten content thereof.

Subsequently, an adsorbent is allowed to adsorb the peel oil. Theadsorbent preferably is a porous adsorbent that is electrically neutraland has a large specific surface, and can be a resin containing acopolymer of styrene and divinylbenzene, for example. Also, consideringincreasing the amount of adsorption to achieve an efficient auraptenpurification, it is preferable that the resin used as the adsorbent isused under dry conditions.

The adsorbent that has adsorbed the peel oil is washed with an alcoholaqueous solution, thereby removing impurities adsorbed to the adsorbent.The alcohol concentration of the alcohol aqueous solution is, forexample, lower than 50% (volume ratio), preferably 10% to 45%, and morepreferably 35% to 45%. The above-noted alcohol can be, for example,ethanol, isopropyl alcohol or the like, and preferably is ethanol whenpurified aurapten is used as food additives and preferably is isopropylalcohol when it is used as drugs. Further, the amount of the alcoholaqueous solution preferably is about 14 times (volume ratio) that of anaurapten-containing solution adsorbed to the adsorbent, for example,though not limited particularly.

Then, aurapten is eluted from the adsorbent using an alcohol aqueoussolution. Since this eluate contains aurapten, it may be used as it isor may be used after being concentrated or dried. Thus obtained auraptensubstantially is white, tasteless and odorless. The alcoholconcentration of the alcohol aqueous solution is, for example, 50% to95%, preferably 60% to 90%, and more preferably 75% to 85%. The alcoholaqueous solution and its amount to be added are similar to thosedescribed above.

Now, examples of the present invention will be described. It should benoted that the present invention is not limited to these examples.

EXAMPLE 1

As described in the following, the present example confirmed theactivation of PPARγ by aurapten.

CV-1 cells (cultured cells derived from kidneys of male African greenmonkeys) were implanted on 24-well culture plates so as to be 0.2μg/well and cultured at 37° C. in 5% CO₂ for 24 hours. As a medium, DMEM(Dulbecco's Modified Eagle Medium; manufactured by GIBCO) containing 10%FBS (fetal bovine serum) and a 10 mg/mL penicillin streptomycin solutionwas used. Using the Lipofectamine system (trade name; manufactured byInvitrogen Corporation), pM-hPPARγ and p4×UASg-tk-luc were transfected.The above-noted pM-hPPARγ was a chimeric protein-expression plasmidbinding GAL4 gene (amino acid sequence 1-147) and human PPARγligand-binding site gene (amino acid sequence 204-505), whereas theabove-noted p4×UASg-tk-luc was a reporter plasmid containing four copiesof a responsive element (UAS) of GAL 4 upstream of a luciferase gene.About 24 hours after the transfection, samples of aurapten at respectiveconcentrations (0.1, 1.0, 10, 50 and 100 μM) were added to theabove-described media, followed by 24 hour incubation. The above-notedsamples were prepared by dissolving aurapten in dimethyl sulfoxide(DMSO). Instead of the aurapten, DMSO was added to non-treated controlmedia. After the incubation, measurement was made using aDual-Luciferase Reporter Gene Assay system (trade name; manufactured byPromega Corporation).

Similarly to the measurement group, as a control group, pM (a plasmidfrom which PPARγ ligand-binding site gene was removed) was used formeasurement instead of pM-hPPARγ. For each sample, the ratio betweenaverage luminescence intensities of the measurement group and thecontrol group (n=4) (measurement group/control group) was calculated,and an activity relative to the non-treatment control was determined asthe PPARγ ligand activity of the sample. Table 1 below and the graph ofFIG. 1 show the results.

TABLE 1 Addition concentration PPARγ ligand activity Non-treatment(0.1%) 100 control (DMSO) Aurapten 0.1 μM 128 ± 8.5  1.0 μM 113 ± 6.9 10 μM 146 ± 17.0 50 μM 245 ± 56.1 100 μM 641 ± 84.2 (average ± standarderror)

As becomes clear from Table 1 and FIG. 1 mentioned above, the auraptenimproved the activity of PPARγ such that the PPARγ activity increasedalong with the concentration of aurapten.

EXAMPLE 2

As described in the following, the present example confirmed theactivation of PPAR_(α) by aurapten.

The PPAR_(α) ligand activity of aurapten was measured similarly toExample 1 except that pM-hPPAR_(α) was used instead of pM-hPPARγ and theaurapten concentrations were set to 0.1, 1.0, 10, 50 and 80 μM. Table 2below and the graph of FIG. 2 show the results.

TABLE 2 Addition concentration PPARα ligand activity Non-treatment(0.01%) 100 control (DMSO) Aurapten 0.1 μM 234 ± 39.4 1.0 μM 139 ± 38.310 μM 302 ± 98.2 50 μM 409 ± 27.8 80 μM 1052 ± 232.9 (average ± standarderror)

As becomes clear from Table 2 and FIG. 2 mentioned above, the auraptenimproved the activity of PPAR_(α) such that the PPAR_(α) activityincreased in keeping with the concentration of aurapten.

EXAMPLE 3

As described in the following, the present example confirmed thepromotion of adiponectin secretion by aurapten.

(Differentiation Induction of Preadipocyte)

First, the following two kinds of media were prepared.

Differentiation Induction Medium (0.25 μM DEX, 0.5 mM MIX, 10 μg/mLInsulin/10% FBS/DMEM)

55 mL of FBS (fetal bovine serum (manufactured by GIBCO)) was added to500 mL of DMEM (manufactured by SIGMA) so as to prepare 10% FBS/DMEM. Tothis 10% FBS/DMEM, 138.75 μL of 1 mM DEX (dexamethasone)/DMSO(manufactured by Nacalai Tesque, Inc.) and 555 μL of 10 mg/mLinsulin/PBS (manufactured by SIGMA) were added. Incidentally, theinsulin/PBS was obtained by adding 1 N HCl to PBS in advance so as tomake the solution acidic enough to allow insulin dissolution and thendissolving insulin. MIX (3-isobutyl-1-methylxanthine) (manufactured byNacalai Tesque, Inc.) was added to a necessary amount of theabove-described medium immediately before use in such a manner as toachieve 0.5 mM, thereby preparing a differentiation induction medium.Since MIX was very difficult to dissolve, it first was dissolved in asmall amount of 99.5% ethanol and then added to 10% FBS/DMEM. At thistime, an adjustment was made so that the final concentration of 99.5%ethanol did not exceed 1%.

Differentiation Promotion Medium (5 μg/mL Insulin/10% FBS/DMEM)

A differentiation promotion medium was prepared by adding 277.5 μL of 10mg/mL insulin/PBS to 555 mL of 10% FBS/DMEM.

Next, culture preadipocytes 3T3-L1 were thawed, inoculated in a 100 mmdish and cultured until 3T3-L1 cells reached about 80% confluence. Asingle dish of 10T½ that had reached about 80% confluence wassubcultured to a single 6-well plate and further cultured until the3T3-L1 cells reached confluence in the 6-well plate, and then the mediumwas replaced with the differentiation induction medium, followed bydifferentiation induction. 48 hours later, the medium was replaced withthe differentiation promotion medium, and thereafter, the medium wasreplaced with the differentiation promotion medium every two days. 7days after the initiation of the differentiation induction, mRNA wasextracted using Sepasol®—RNA I super (manufactured by Nacalai Tesque,Inc.), and the mRNA expression amounts of 36B4, aP2 and adiponectin,which were indicators of an early period of adipocyte differentiation,were measured using Light Cycler™. Also, the medium 7 days after thedifferentiation induction was taken in an amount of 1 mL from each well,and the amount of adiponectin in the medium supernatant was measuredusing a mouse/rat adiponectin ELISA kit (trade name) (manufactured byOtsuka Pharmaceutical Co., Ltd.).

(Quantitative Determination of mRNA Using Light Cycler™

Extraction and Quantitative Determination of Total RNA

The medium was removed from the above-described 6-well plate, 1 mL ofSepasol®—RNA I Super (manufactured by Nacalai Tesque, Inc.) was added toeach well, and pipetting was repeated several times so as to dispersethe cells. This solution was transferred to a 1.5 mL tube and allowed tostand for 5 minutes at room temperature, and then 200 μL of chloroformwas added, stirred well with a vortex stirrer and allowed to stand for 3minutes at room temperature. The solution was cooled to 4° C. andcentrifuged at 12000×g for 15 minutes. While taking great care not todisturb an interface between a phenol layer (a lower layer, yellow) anda water layer (an upper layer, colorless), the water layer alone wastransferred to another tube (with a capacity of 1.5 mL). At this time,care was taken not to take proteins floating between these layers. 500μL of isopropanol was added to the tube and mixed and allowed to standfor 10 minutes at room temperature. The solution was cooled to 4° C. andcentrifuged at 12000×g for 10 minutes, followed by removing about 1 mLof the supernatant. To this precipitate, 1 mL of 75% ethanol was addedand stirred to suspend the precipitate sufficiently, and then cooled to4° C. and centrifuged at 12000×g for 10 minutes, followed by removingthe supernatant. The resultant precipitate (total RNA) was dried andthen dissolved in 20 μL of nuclease free water, thus measuring theconcentration of mRNA using Nanoprop (manufactured by SCRUM Inc.).

Reverse Transcription

The extracted and measured mRNA solution was adjusted to have an mRNAconcentration of 1 μg/1 μL of Oligo dT primer and 10 μL of theabove-described RNA solution were added to 8-tube strips (with acapacity of 0.2 mL). The mixture solution was incubated in a Thermalcycler at 70° C. for 10 minutes so as to destroy higher-order structuresof RNA, and transferred onto the ice and allowed to stand for at least 1minute. Then, 11 μL of RNA sample/primer mixture solution, 5 μL of5×reverse transcription buffer, 1 μL of RNase inhibitor, 5 μL of 2.5 mMdNTP Mix and 2 μL of Nuclease Free water were added in this order (24 μLin total).

After pre-incubation at 42° C. for 5 minutes in the Thermal cycler, 1 μLof reverse transcriptase was added, and the content of the tube wasmixed well by pipetting. After incubation at 42° C. for 50 minutes andfurther incubation at 70° C. for 15 minutes in the Thermal cycler, thecontent was cooled on the ice and centrifuged mildly so as to collectthe reaction solution to the tube bottom, and then frozen and stored at−20° C. Every time it was used for the Light Cycler™ measurement, it wasdiluted by 10 times.

Measurement Using Light Cycler™

All the operations described below were carried out in a clean bench. 5mL of a plasmid solution containing fragments of the gene whoseexpression amount was to be measured was poured in a 0.65 mL tube anddiluted by 10 times with 45 mL of water attached to a Light Cycler™ DNAMaster SYBR Green (trade name). By repeating these operations, 10²-time,10³-time, 10⁴-time, 10⁵-time, 10⁶-time, 10⁷-time and 10⁸-time dilutedsolutions were produced, respectively. A dedicated capillary was setinto a Light Cycler™ Centrifuge Adapter (trade name) using tweezers, and18 μL each of the above-noted reagent was dispensed thereto. Further, 2μL of water (negative control) or the 7-step diluted solution (standard)and 2 μL of 10-time diluent of cDNA serving as a measurement sample wereadded, and a lid was placed using tweezers. After centrifugation at 5000rpm at 4° C. for 10 seconds, the capillary was mounted on a carousel,which then was set in a chamber and measured.

(Measurement of Adiponectin Secretion Amount Using ELISA)

The above-mentioned mouse/rat adiponectin ELISA kit had the followingconfiguration.

Stock solution for washing Stock solution for diluting analyte Antibodyplate (anti mouse adiponectin polyclonal antibody (rabbit) solid phaseplate)

8.0 ng/mL standard product (recombinant mouse adiponectin)

Biotin labeled antibody solution (biotin labeled anti mouse adiponectinpolyclonal antibody (rabbit)) Enzyme-labeled streptavidin stock solution(HRP-labeled streptavidin) Enzyme-labeled streptavidin diluent Substratesolution A (3,3′,5,5′-tetramethylbenzidine) Substrate solution B(hydrogen peroxide) Reaction stop solution

First, the following reagents and analyte solution were prepared.

Washing Solution

The above-noted stock solution for washing and purified water were mixedin a ratio of 40 mL to 960 mL and stored at 2.8° C.

Analyte Diluent

The above-noted stock solution for diluting analyte and purified waterwere mixed in a ratio of 50 mL to 200 mL and stored at 2.8° C.

Standard Solution

The above-noted 8.0 ng/mL standard product was diluted in two steps withthe above-described analyte diluent, thus preparing standard solutionshaving a concentration of 4.0 ng/mL, 2.0 ng/mL, 1.0 ng/mL, 0.5 ng/mL and0.25 ng/mL.

Enzyme-Labeled Streptavidin Solution

The above-noted enzyme-labeled streptavidin diluent and the above-notedenzyme-labeled streptavidin stock solution were mixed in a ratio of 12mL to 60 μL.

Substrate Solution

The above-noted substrate solution B and the above-noted substratesolution A were mixed in a ratio of 6 mL to 6 mL.

Analyte Solution

Using the above-described analyte diluent, the supernatants of culturesto which control and ligand candidate were added respectively werediluted by 25 times, and the supernatant of a culture to whichpioglitazone was added as positive control was diluted by 50 times.

Only the strips of the antibody plates necessary for the analysis weretaken out. After about 200 μL of the above-described washing solutionwas poured to each well of the antibody plate, the liquid in the wellwas absorbed and removed completely using a plate washer. This washingand absorption were performed once more. 100 μL of the standardsolutions with respective concentrations and 100 μL of the dilutedanalytes were added to individual wells and measured in duplicate.Incidentally, the standard solutions always were measured for eachmeasurement and each plate. After the antibody plate was covered with aplate seal and allowed to stand still for a reaction for 60 minutes atroom temperature, the plate seal was removed from the antibody plate,followed by absorbing and removing completely the liquid in the wellusing the plate washer. Subsequently, about 200 μL of the washingsolution was added to each well and immediately was absorbed andremoved. This washing and absorption were repeated four more times.After 100 μL of the biotin labeled antibody solution was added to eachwell of the antibody plate, the antibody plate was covered with a plateseal and allowed to stand still for reaction for 60 minutes at roomtemperature. Similarly to the above, the washing of the wells andabsorption were repeated five times. After 100 μL of the enzyme-labeledstreptavidin solution was added to each well of the antibody plate, theantibody plate was covered with a plate seal and allowed to stand stillfor reaction for 60 minutes at room temperature. Similarly to the above,the washing of the wells and absorption were repeated five times. After100 μL of the substrate solution was added to each well of the antibodyplate and allowed to stand still for reaction for 15 minutes at roomtemperature, 100 μL of the reaction stop solution was added to each wellof the antibody plate, and then the absorbance at 450 nm in each wellwas measured with a plate reader.

Using the results of quantitative determination with the Light Cycler™,the ratio of the respective mRNA expression amounts of aP2 andadiponectin with respect to the mRNA expression amount of 36B4 wascalculated for each sample. The results of the calculation are shown inTable 3 below and the graph in FIG. 3. Also, the measurement results ofthe adiponectin secretion amount using ELISA are shown in Table 4 belowand the graph in FIG. 4.

TABLE 3 Addition concentration aP2 Adiponectin Non-treatment   100 ±12.4   100 ± 12.4 control Aurapten 1 μM 208.2 ± 23.4 144.5 ± 26.7 5 μM202.7 ± 19.3 161.4 ± 40.2 (average ± standard error)

TABLE 4 Addition concentration Secretion amount Non-treatment 3.49 ±0.78 control Aurapten 1 μM 3.64 ± 0.82 5 μM 4.69 ± 0.16 (average ±standard error)

When adipocytes cultured in the differentiation induction medium towhich aurapten was added and adipocytes cultured in the non-treatedcontrol medium were compared, the addition of aurapten was found toenhance the secretion of adiponectin in the adipocytes.

EXAMPLE 4

As described in the following, the present example confirmed theinhibition of VLDL secretion by aurapten.

(Cell Culture)

FCS, a non-essential amino acid, sodium pyruvate and a glutaminesolution were mixed such that the final concentrations of the FCS, thenon-essential amino acid, the sodium pyruvate and the glutamine solutionrespectively were 10%, 1%, 1 mM and 2 mM, and added to an MEM medium(manufactured by SIGMA). They were mixed aseptically on a clean bench.In a 100 mm/Collagen-Coated Dish (trade name, manufactured by IwakiGlass Works, Co.), the medium of HepG2 cells (human hepatic cells) thathad been cultured to 80% to 90% confluence was removed with a pipetteand washed with 2 mL of 1×PBS. After adding 2 mL of trypsin-EDTA androtating the above-noted dish slowly so that trypsin-EDTA spread overthe entire cells, this trypsin-EDTA was removed with a pipette. The dishwas allowed to stand still for 15 minutes in a CO₂ incubator (37° C.,5%), and then 4 mL of a growth medium was added to this dish, followedby mixing by pipetting. Then, 2 mL of this mixture was added to each oftwo dishes to which 3 mL of a growth medium was added in advance. Thesedishes were covered with lids and moved crisscross so as to mix thecontent. The cells were checked using a microscope (manufactured byOlympus Corporation) and cultured in a CO₂ incubator (37° C., 5%). Threeor four days later, after checking that 80% to 90% confluence wasachieved using the microscope, subculture was carried out in a similarmanner, followed by cell culture.

1. Time Course Experiment

HepG2 was cultured to reach 80% to 90% confluence, and the medium wasremoved with a pipette, followed by washing with 2 mL of 1×PBS andaddition of 5 mL of a growth medium. Every 10 hours, 200 μL of themedium was collected and put into a tube (with a capacity of 1.5 mL).Using this sample, apoB100 was subjected to Western blotting and ELISAmeasurement.

(Western Blotting)

(1) SDS-PAGE

20 μL of the medium and a buffer containing 62.5 mM Tris-HCl (pH 6.8),2% SDS, 10% glycerol, 5% (w/v)₂-mercaptoethanol and a 0.0005% BPBsolution were put in a 1.5 mL tube so as to achieve the total amount of30 μL, and stirred well. They were boiled in a hot water bath at 100° C.for 5 minutes. An acrylamide gel containing 7.5% SDS was set in MiniPROTEAN3 Cell (trade name, manufactured by Bio-Rad Laboratories, Inc.),and 300 μL of an electrophoresis buffer was poured so that the gel wassoaked sufficiently. The electrophoresis buffer was prepared by diluting30 mL of 10×Tris/Glycine/SDS buffer (manufactured by Bio-RadLaboratories, Inc.) with 270 mL of dH₂O. Then, 30 μL of the sample and 5mL of Rainbow Marker (trade name, manufactured by Promega Corporation)were dispensed quietly into the gel so as to perform electrophoresis.The condition of electrophoresis was a constant voltage of 200 V and aperiod of 40 to 45 minutes. Model 3000xi Computer ControlledElectrophoresis Power Supply (trade name, manufactured by Bio-RadLaboratories, Inc.) was used as the power supply.

(2) Blotting

The gel that had been subjected to SDS-PAGE was soaked in atranscription buffer (2.42 mg/mL Tris base, 11.55 mg/mL Glycine, 20%methanol) for 15 minutes together with a PVDF membrane (Hybond TM-P PVDFtransfer membrane; manufactured by Amersham Biosciences K.K.) untilequilibrium was reached. Using a semidry flat transfer apparatus(manufactured by NIHON EIDO Co., Ltd.), the transcription was performedonto the PVDF membrane by a semidry method (40 mA/membrane, 90 minutes).This membrane was blocked with 5% skim milk (at room temperature for 1hour). After the blocking, 5 mL of Ms×Hu Apolipoprotein B (manufacturedby CHEMICON International, Inc.) that had been diluted with 5% skim milkby 1000 times was dispensed to the above-noted membrane and allowed toreact at room temperature for 1 hour. The resultant membrane was washedwith PBST three times (10 minutes, 20 minutes, 30 minutes), and 5 mL ofanti mouse IgG-HRP (manufactured by Promega Corporation) that had beendiluted with 5% skim milk by 5000 times was poured uniformly to theabove-noted membrane and allowed to react at room temperature for 1hour. The membrane was washed with PBST three times (10 minutes, 50minutes, 10 minutes), and the detection was carried out by achemiluminescence method using ECL+plus western blotting detectionsystem (trade name, manufactured by Amersham Biosciences K.K).

(Elisa)

First, a VLDL standard solution was prepared. The VLDL standard solutionwas prepared by diluting 1.169 mg/mL of human VLDL standard (trade name,manufactured by CHEMICON International, Inc.) with a growth medium. Thedilution factors were 100, 1000, 10000, 100000 and 1000000.

A sandwich ELISA that recognized human apoB10 in the VLDL as an antigenwas used. First, 100 μL of Moab×LDL Apolipoprotein B (ApoB)(manufactured under MONOSAN®) was dispensed in each well of ELISA PLATE,and the plate was sealed with a sterile seal and then allowed to standstill at 4° C. overnight. In the following, all the dispensing mentionedis for a single well. On the next day, 200 μL of Zepto Block (tradename, manufactured by ZeptoMetrix Corporation) was dispensed, and thenthe above-noted plate was sealed and allowed to stand still for 2 hoursat room temperature for blocking. Subsequently, 100 μL of the medium andthe VLDL standard solution were dispensed, and then the above-notedplate was sealed and allowed to stand still for 1 hour at roomtemperature. Washing with 200 μL of PBST and absorption using anaspirator were carried out five times in total. Then, 100 μL of AffinityPurified Anti-Apolipoprotein B (manufactured by RocklandImmunochemicals, Inc.) that had been diluted with PBS by 1000 times wasdispensed and allowed to stand still for 1 hour at room temperature.After washing similarly to the above, 100 μL of Donkey Anti-goat IgG HRP(manufactured by Promega Corporation) that had been diluted with PBS by5000 times was dispensed and allowed to stand still for 1 hour at roomtemperature. After washing similarly to the above, solutions A and B ofTMB Microwell Peroxidase substrate (trade name, manufactured byFunakoshi Co., Ltd.) were mixed and allowed to stand still for 5 minutesat room temperature, and then 100 μL of this mixture solution wasdispensed and allowed to stand still for 5 minutes at room temperature.100 μL of 1M phosphoric acid solution was added to the above-notedmixture solution, and the absorbance at 450 nm was measured using wallacARVOsx (trade name, PerkinElmer Life Sciences, PerkinElmer, Inc.). Themode of measurement was Photometry (450 nm, 1.0 S).

From the result of the Western blotting of human apoB100, a clear bandwas shown 20 hours or more after the start of culture. Also, from theresult of the ELISA, a linear increase in the VLDL secretion amount wasshown from the initiation of culture to about 50 hours thereafter. Inview of these results, a collection time of the medium in a VLDLsecretion inhibition experiment, which will be described below, was setto 30 hours.

2. VLDL Secretion Inhibition Experiment

(Preparation of Medium Containing Aurapten)

Into 15 mL centrifugation tubes, 3 mL of a medium was added, andfurther, aurapten was added so as to achieve final concentrations of 10μM, 20 μM and 50 μM. They were tumble-mixed to a sufficient degree, thuspreparing media containing aurapten. Also, as a control, a medium wasprepared by adding the same amount of DMSO instead of aurapten.

HepG2 was cultured to 80% to 90% confluence in a 100 mm/Collagen-CoatedDish (trade name, manufactured by Iwaki Glass Works, Co.). The mediumwas removed from the above-noted dish with a pipette and then HepG2 waswashed with 2 mL of 1×PBS. Then, 2 mL of trypsin-EDTA was added, theabove-noted dish was rotated slowly so that the trypsin-EDTA spread overthe entire cells, this trypsin-EDTA was removed with a pipette, and thedish was allowed to stand still for 15 minutes in a CO₂ incubator (37°C., 5%). To this dish, 12 mL of a growth medium was added, followed bymixing by pipetting, and 1 mL of this mixture solution was dispensed ineach well of Collagen-Coated Microplates 12Well/Flat Bottom (trade name,manufactured by Iwaki Glass Works, Co.). After the cells were checkedusing a microscope, the wells were cultured in a CO₂ incubator (37° C.,5%) for one to two days. After checking that 80% to 90% confluence wasachieved using the microscope, a medium replacement was carried out byremoving the medium and adding 800 μL of a medium containing aurapten.Thirty hours later, 800 μL of the medium was collected from each well.Using this collected medium, the number of living cells were counted,and the Western blotting and the ELISA measurement were carried outsimilarly to the above. From the result of the ELISA, the ratio of theVLDL secretion amount between the measurement group and the non-treatedcontrol group (measurement group/control group) was calculated for eachsample.

(Counting of the Number of Living Cells)

The measurement was made using CellTiter 96 Aqueous One Solution CellProliferation Assay (trade name, manufactured by Promega Corporation).First, 1.6 mL of CellTiter 96 Aqueous One Solution Reagent and 6.4 mL ofa growth medium were added to a centrifugation tube (with a capacity of15 mL) and stirred well. Then, 600 μL of the mixture solution was addedto each well immediately after the medium was stirred, and incubated for40 minutes in a CO₂ incubator (37° C., 5%). 100 μL of this mixturesolution was dispensed to each of three wells of ELISA PLATE 96well(trade name, manufactured by Iwaki Glass Works, Co.), and the absorbanceat 490 nm was measured. The measurement was made using wallac ARVOsx(trade name, PerkinElmer Life Sciences, PerkinElmer, Inc.), and the modeof measurement was Absorbance (490 nm, 1.0 S). The ratio of the numberof living cells between the measurement group and the non-treatedcontrol group (measurement group/control group) was calculated for eachsample.

The results are shown in Table 5 below and the graph in FIG. 5.

TABLE 5 Addition VLDL secretion Number of concentration ratio (%) livingcells (%) Non-treatment 100 100 control (DMSO) Aurapten 10 μM 63.1 ±5.31 100.3 20 μM 61.6 ± 2.21 99.9 50 μM 45.6 ± 4.04 98.7 (average ±standard error)

As becomes clear from the results above, the VLDL secretion wasinhibited by aurapten, and the degree of inhibition increased further asthe aurapten concentration rose.

INDUSTRIAL APPLICABILITY

As described above, since the metabolic syndrome relieving agentaccording to the present invention has an excellent PPAR_(α) activityand PPARγ activity and a function of promoting adiponectin secretion, itis extremely effective in relieving a metabolic syndrome and can be usedas a drug, a supplement, a functional food and a food additive forpreventing and treating diseases such as insulin resistance,hyperinsulinism, type 2 diabetes, hypertension, hyperlipemia,arteriosclerosis, obesity and visceral fat obesity, for example. Itshould be noted that they are effective for not only humans but alsoother animals.

1. An agent for relieving a metabolic syndrome, comprising aurapten. 2.The agent according to claim 1, wherein the metabolic syndrome comprisesat least one disease selected from the group consisting of insulinresistance, hyperinsulinism, type 2 diabetes, hyperlipemia,arteriosclerosis, hypertension, obesity and visceral fat obesity.
 3. Theagent according to claim 1, which activates a peroxisomeproliferator-activated receptor (PPAR).
 4. The agent according to claim1, wherein the PPAR is at least one of a PPARα and a PPARγ.
 5. The agentaccording to claim 1, wherein the aurapten is derived from at least oneselected from the group consisting of a fruit, a fruit juice and a peelof a citrus fruit.
 6. The agent according to claim 5, wherein the citrusfruit is selected from the group consisting of a sweet summer orange, ahassaku orange, a Watson pomelo and a grapefruit.
 7. A drug forpreventing or treating a metabolic syndrome, the drug comprising theagent according to claim
 1. 8. A supplement for preventing or relievinga metabolic syndrome, the supplement comprising the agent according toclaim
 1. 9. A functional food for preventing or relieving a metabolicsyndrome, the functional food comprising the agent according to claim 1.10. A food additive for preventing or relieving a metabolic syndrome,the food additive comprising the agent according to claim
 1. 11. A PPARactivator comprising aurapten.
 12. An adiponectin secretagoguecomprising aurapten. 13-15. (canceled)
 16. A method for relieving ametabolic syndrome, comprising administering aurapten to a mammal.
 17. APPAR activating method comprising activating a PPAR with aurapten.